The present invention is in the field of disease detection and therapy. The present invention specifically provides the identification of previously unknown nucleic acid/amino acid polymorphisms within the estrogen receptor beta gene (ESR-beta) and the genomic sequence of this gene for use in the development of diagnostics and therapies for diseases and disorders mediated/modulated by the estrogen receptor.
Estrogen Receptor
The human estrogen receptor beta belongs to the nuclear hormone receptor family. Nuclear hormone receptors are a family of hormone-activated transcription factors that can initiate or enhance the transcription of genes containing specific hormone response elements.
The ER protein consists of 595 amino acids with a molecular weight of 66 kDa, 8 transcribed exons, with six different functional domains. Two of those domains are highly conserved in the primary sequence of members of the nuclear hormone receptor superfamily. One of the domains, the DNA binding domain (DBD), contains two zinc fingers that mediate receptor binding to hormone response elements in the promoters of hormone-responsive genes. In the C-terminal region, the hormone-binding domain (HBD) contains two regions of sequence homology with other hormone receptors and gives hormone specificity and selectivity. The human ER-alpha gene is located in chromosome 6q.25.1.Estrogen receptors, like other steroid receptors, are transcription factors that are activated upon binding to steroids (estradiol) or steroid analogs such as tamoxifen. Upon activation the receptors dimerize to form homodimers or heterodimers that bind to estrogen receptor elements (EREs) located in the promoter region of estrogen-activated genes and coordinate transcription by interacting with host co-activators.
Role of Estrogen in Cardiovascular Disease
Heart disease is the leading cause of mortality in women, a fact that is under appreciated by both women and physicians. One in 9 women aged 45-65 have some form of cardiovascular disease and the number increases to 1 in 3 after age 65. Each year, 240,000 U.S. women die from heart disease, and nearly 90,000 die of stroke. Moreover, approximately 44% die within one year of suffering a heart attack, compared with 26% of men (Warren M P and Kulak J Clin Obs Gyn 1998 41(4):976-987).
Estrogens exert a wide range of physiological effects on a large variety of cell types. For example, they regulate cell growth and apoptosis and a myriad of functions related to reproduction. There are two types of estrogen receptors, beta and beta. Blood vessels and bone contain beta receptors, the liver has beta receptors, and both beta and beta receptors are found in the central nervous system. The interaction of these different receptor sites influences the biological effects of estrogen and selective estrogen receptor modulators (SERMs), such as raloxifene. The binding patterns dictate whether an estrogen or a SERM acts as an estrogen agonist or an antagonist (Mendelsohn ME and Karas R H New Engl J Med 1999, 340(23):1801-1811; Grese T A and Dodge J A Curr Pharm Design 1998, 4:71-92). Tissue-specific relationships exist between SERMs and the receptor binding sites. Estrogens also increase high-density lipoprotein cholesterol levels, decrease low-density lipoprotein cholesterol, and decrease plasminogen-activating inhibitor levels (Meisler J G Jour Women""s Health 1999, 8(1):51-57). All estrogens require cellular receptors for their expression. In general, estrogen receptors are ligand-inducible transcription factors, which regulate the expression of target genes after hormone binding (Faustini-Fustini et al. Eur J Endocrin 1999, 140:111-129). Estrogen may also have important effects on the vascular wall. Estradiol and progesterone receptors have been identified in arterial endothelial and smooth muscle cells (Campisi D et al. Int J Tiss React 1987, IX(5):393-398). Estrogens act on the wall of the artery to relax vascular smooth muscle and to decrease vascular resistance. The mechanism appears to be through stimulation of endothelial-derived relaxing factors and an endogenous nitrate (Warren M P and Kulak J Clin Obs Gyn 1998 41(4):976-987). The relaxation induced by 17B-estradiol may play an important role in the regulation of coronary tone, which reduces the risk of coronary disease in postmenopausal women. The production of nitric oxide is mediated by the estrogen receptor, because when the receptor is blocked by an antiestrogen agent, nitric oxide is suppressed.
Several studies have shown that estrogen therapy reduces the risk of heart disease by up to 50% (most recently reviewed by Mendelsohn M E and Karas R H New Engl J Med 1999, 340(23):1801-1811; Rich-Edwards J W N Engl J Med, 1995, 332:1758-1765; Gerhard M, Ganz P, Circulation, 1995, 92:5-8; Grodstein F, et al N Engl J Med 1997, 336:1769-75; Chasen-Taber L and Stampfer M J Ann of Int Med, 1998, 128:467-477; Warren M P and Kulak J Clin Obstet Gyn 1998, 41(4):976-987). Loss of estrogen may be one of the most important factors in the development of cardiovascular disease in women.
While there is no direct evidence that estrogen prevents atherogenesis, considerable epidemiologic evidence exists that suggests that estrogens may have some benefit in reducing cardiovascular disease: (1) In all age groups, women have a lower incidence of cardiovascular disease than do men; (2) women who undergo a premature surgical menopause and do not take estrogens are twice as likely to have cardiovascular disease are age-matched premenopausal controls; (3) postmenopausal women who use estrogens have a significantly lower incidence of cardiovascular disease compared with those who do not; and (4) women with coronary artery disease detected by angiography have a higher survival rate if they are estrogen users.
In recent years, reports of favorable effects of estrogen therapy on cardiovascular morbidity and mortality have led to enthusiasm for widespread use of estrogens by postmenopausal women (Meinertz T Herz 1997, 22: 151-157). Guidelines for estrogen therapy issued by the American College of Physicians include the statement xe2x80x9cWomen who have coronary heart disease are likely to benefit from hormone therapy.xe2x80x9d
More than 30 prospective studies and 13 case controlled studies have examined the effect of estrogen replacement therapy on cardiovascular incidence or prevalence and all cause mortality (Stampfer M J et al. New Engl J Med 1991, 325:756-62; Grady D et al. Ann Intern Med 1992, 117:1016-37). The majority of these studies showed lower morbidity and mortality from coronary heart disease among users of postmenopausal estrogens than among non-users. Specifically, they have shown that coronary artery disease in estrogen takers is approximately 50% that in women who do not take estrogen. Overall, the bulk of the evidence strongly supports a protective effect of estrogens yielding a relative risk of 0.56 (95% confidence interval 0.50-0.61). However, a xe2x80x9chealthy woman selection biasxe2x80x9d is present in these studies and potentially may confound these results (estrogen takers have better weight control, exercise more, and smoke less than women who are not prescribed estrogen). Moreover, other biases such as estrogen takers tend to have higher education, higher income, etc., are confounding these epidemiologic studies (Abrams J Clin Cardiol 1998, 21:218-222).
Since the earlier observational trials were not randomized, it is believed by many that as much as 25% of this 50% reduction in risk is due to these various methodological biases (Barrett-Conner E and Grady D 1998, Ann Rev Public Health 19:55-72). Recently, 2 meta-analyses estimated the reduction in coronary heart disease associated with estrogen use to be in the range of 35 to 44%, respectively (Grodstein F and Stampfer M J Prog Cardiol Dis 1995, 38: 199-210; Barrett-Conner E and Grady D 1998, Annu Rev Public Health 19:55-72). Recent studies are exploring the issue of opposed vs unopposed estrogen, because of a documented increased risk for uterine cancer in women with an intact uterus who are taking estrogen alone. The new lines of evidence are suggesting that women taking estrogen plus a progestin (usually a medroxyprogesterone acetate) do not receive an equivalent benefit from the cardioprotective effects compared to women taking estrogen alone (Hulley S et al 1998 JAMA 280:605-613; Abrams J Clin Cardiol 1998, 21:218-222).
The loss of estrogen at menopause is associated with a 6% decline in HDL cholesterol levels and a 5% rise in LDL cholesterol levels, which may explain the higher cardiovascular disease rate among postmenopausal women compared with premenopausal women. The lower incidence of cardiovascular disease among postmenopausal women who take estrogen may be explained in part by the resultant 15% to 19% decrease in LDL cholesterol levels and the 16% to 18% increase in HDL cholesterol levels (JAMA 1995, 273:199-208). The PEPI (Postmenopausal Estrogen/Progestin Intervention, a randomized, double-blind placebo-controlled trial, showed that HDL cholesterol levels rose significantly more in women assigned to estrogen alone than in women assigned the combined estrogen (JAMA 1995, 273:199-208). Recent non-human primates studies substantiate these findings (Clarkson T B Lab An Sci 1998, 48(6):569-72). Statistical modeling of the effect of estrogen on lipid profiles indicates that 25-50% of the apparent cardioprotection due to estrogen is mediated by favorable changes in HDL-cholesterol (Bush T L et al. 1987 Circulation 75:1102-9; Gruchow H W et al. 1988 Am Heart J 115:954-63).
Estrogen replacement therapy is not without risk. For years, studies have shown a 3-4-fold increased risk of venous thromboembolism (VTE) in users of oral contraceptives compared to non-users (Weiss G Am J Obstet Gynecol 1999 180:S295-301). One study has shown that intrinsic coagulation factors play a significant role in oral contraceptive-associated VTE (Vandenbroucke J P et al. Lancet 1994 344:1453-7; Rosing J et al. Br J Haematol 1997, 97:233-238). The Factor V Leiden mutation increases risk of VTE 5-10 fold in non users, but 30-fold in third-generation oral contraceptive users. Combined estrogens appear to induce resistance to the body""s natural anticoagulation system (APC). Heterozygotes for the Factor V Leiden mutation who take oral contraceptives develop APC resistance as high as that seen in women who are homozygous.
Estrogens increase the risk of endometrial carcinoma approximately 6-fold, an effect that is eliminated, for the most part, by the addition of progestins (Barrett-Conner E and Grady D 1998, Ann Rev Public Health 19:55-72). Controversy continues over whether estrogen replacement increases the risk of breast cancer, but some studies indicate risk is elevated by as much as 30%. (Greendale GA et al. Lancet 1999, 353:571-80).
A number of prospective randomized studies designed to definitely establish whether estrogen replacement therapy reduces the risk of cardiovascular disease in women and whether it increases the risk of breast cancer, are underway. One recently completed trial (HERSxe2x80x94Heart and Estrogen/progestin Replacement Study) compared continuous combined estrogen plus medroxyprogesterone acetate to placebo in 2700 women with pre-existing coronary disease (Hully S et al. 1998 JAMA 280(7):605-13). Compared to controls, the intervention group had significantly more heart disease events in year one of the trial, but significantly fewer events in years 4 and 5 of the trial. Moreover, a significant increase in the rate of thromboembolic events occurred in the early years of the study in women taking hormones. Based on these results, hormone replacement therapy is not recommended for secondary prevention of heart disease.
Two other large, ongoing clinical trials on primary prevention of cardiovascular disease using estrogens are underway. The Women""s Health Initiative, due to be completed in 2005 and a U.K study called WIS-DOM, due to be completed in 2010, should shed new light on the protective effects of estrogen on cardiovascular disease (Meisler J G Jour Women""s Health 1999, 8(1):51-5).
In summary, ongoing research suggests that estrogen replacement therapy, particularly involving recently formulated designer estrogens or SERMs, may have beneficial effects on the cardiovascular system as well as bone, without the untoward effects on breast and endometrial tissue. Caution still needs to be observed, nonetheless. Women who take estrogens are, on average, better educated, healthier, have higher incomes and have better access to health care. These differences rather than the estrogens may account for much of the lower risk of heart disease.
For postmenopausal women without frank disease, estrogen replacement therapy appears to have a beneficial effect when one considers the magnitude, consistency, and biological plausibility of the data. For women with pre-existing disease, questions remain as to the safety and efficacy of exogenous estrogens as protective agents against cardiovascular disease.
Estrogen and Autoimmune Diseases
A. Systemic Lupus Erythematosus
There is a widely held view that estrogens play a role in Systemic lupus erythematosus because:
1. Women of child bearing age are nine times more likely to develop systemic lupus erythematosus than men. Prior to pubescence the rate is three fold higher in females, while post menopausal women have an equal chance of developing SLE as aged matched males. Many studies have been done that show that the reason for the differences in the sexes is probably estrogen related (Lahita R. G., 1986: Springer Seminars in Immunopathology 9, 305-314; Krammer, G. M. and Tsokos, G. C., 1998 Clinical Immunology and Immunopathology 89: 192-195; Rider at al., 1998 Clinical Immunology and Immunopathology 89: 171-180).
Clues to the role of estrogens in SLE came from studies that concluded that oral contraceptives adversely affected the morbidity of this illness (Buton, J. P., 1996 Ann. Med. Interne, 147:259-264; Julkunen, 1991: Scan. J. Rheumatol. 20:427-433).
2. Patients with Klinefelter syndrome (XXY), have been reported with SLE (Stern et al., 1977: Arthritis and Rheumatism 20:18-22).
3. Patients with SLE have anti-estrogen antibodies (Feldman, 1987: Biochem. Biophys. Acta, 145:1342-1348: Bucala et al., 1987: Clin. Exp. Immunol. 67:167-175)
In the past, oral contraceptives have been shown to cause flare ups of SLE, their use was discouraged in women with SLE, while the current thinking is that the lower dose birth control pills are safe for SLE patients (Julkunen H A Scand J Rheumatol 1991;20(6):427-33). As well hormone replacement therapy is considered safe for SLE patients (Mok et al., Scand J Rheumatol 1998;27(5):342-6: Kreidstein et al., 1997, J Rheumatol 1997 November;24(11):2149-52)
4. The estrogen antagonist tamoxofin seems to improve the course of the disease (Sthoeger, 1997, Ann NY Acad Sci 1997 Apr. 5;815:367-8: Sthoeger, 1994, J Rheumatol 1994 December;21(12):2231-8).
B. Estrogen, Rheumatoid Arthritis (RA) and Osteoarthritis
The literature surrounding the involvement of estrogens in Rheumatoid arthritis is less clear than with osteoarthritis. Epidemiological studies suggests that RA is influenced by female sex hormones, by one study states that the use of oral contraceptives may postpone the onset of RA, but that estrogens alone no not alleviate the symptoms of RA (Bijlsma Am J Reprod Immunol 1992 October-December;28(3-4):231-4). Adjuvant oestrogen treatment does increase bone mineral density in postmenopausal women with RA, and may protect against osteophoresis which is often a complication of RA (van den Brink: Ann Rheum Dis 1993 April;52(4):302-5). While the study mentioned above indicated that estrogens did not alleviate RA symptoms, another study concluded that adjuvant estrogen therapy did not even improve the symptoms. One polymorphism has been reported in the estrogen receptor that seems to be associated with the age of onset of RA (Ushiyama Ann Rheum Dis 1999 January;58(1):7-10)
Osteoarthritis on the other hand is less prevalent in postmenopausal women who take estrogen replacement therapy (ERT) (Felson Curr Opin Rheumatol 1998 May;10(3):269-72) suggesting that ERT may be beneficial in preventing osteoarthritis.
C. Estrogen and Osteophorosis
Osteophorosis is a metabolic bone disorder that leads to bone fragility and subsequent risk of fracture. Treatment for postmenopausal women with osteophoresis includes hormone replacement, in particular estrogen. Estrogen has shown to reduce the incidence of bone loss and fractures (Weiss et al., N Engl J Med 1980 Nov. 20;303(21):1195-8:Paganini-Hill et al., Ann Intern Med 1981 July;95(1):28-31: Ettinger et al., Ann Intern Med 1985 March;102(3):319-24)
Further, polymorphisms in the estrogen receptor have been associated with bone loss in both humans and mice. (Kobayashi J Bone Miner Res 1996 March;11(3):306-11: Kurabayashi Am J Obstet Gynecol 1999 May; 180(5):1115-20; Deng Hum Genet 1998 November;103(5):576-85)
Estrogens and Cognitive Function
Compared with men, women are at greater risk of developing Alzheimer""s disease. Several studies show that women who take estrogen after menopause have a lower incidence of Alzheimer""s disease. Among women with Alzheimer""s, those taking estrogen suffer less severe symptoms and slower mental deterioration. The duration of estrogen use also seems to be important in reducing risk. Women with a history of long-term term use (more than 10 years) had the lowest risk. But even women who took estrogen for a short time also benefited.
Estrogen and Breast Cancer
The major risk factors for the development of breast cancer are sex, age, family history of breast cancer, age of menarche, age at first full-term pregnancy, and age of menopause. All of these factors, with the exception of family history, have been shown to be directly associated with lifetime exposure to estrogen, increased hormone exposure being associated with increased risk of developing breast cancer. The increased cancer risk is believed to be caused by an estrogen receptor-mediated proliferative response in cells of the mammary epithelium.
Tamoxifen, an estrogen receptor antagonist, has been shown to be an effective agent for both the prevention and treatment of breast cancer. Using immunohistochemical methods, it is possible to classify breast tumors as being estrogen receptor positive or negative, depending upon the amount of estrogen receptor protein expressed in the tissue. Estrogen receptor positive tumors are more likely to respond to treatment with tamoxifen than estrogen receptor negative tumors. Pre-menopausal women are more likely to develop estrogen receptor negative breast cancers than are post-menopausal women.
Mutations altering the structure and function of the estrogen receptor have been described in primary breast tumors or breast cancer cell lines. It is not clear however whether these changes are primary (and involved in the processes leading to carcinogenesis) or secondary (and a consequence of genetic instability in cancer tissues). In addition to these somatic mutations, some studies have pointed to a possible association between inherited DNA sequence changes and the development of breast cancer, but these studies are also controversial.
Further evidence for the role of estrogen receptors in breast cancer comes from the recent finding that the gene BRCA 1, which when inherited in a mutant form predisposes to the development of breast cancer, inhibits estrogen receptor signaling.
Estrogens and Endometrial Cancer
Carcinoma of the endometrium is the most common pelvic malignancy in women, however because in approximately 75% of cases it is confined to the body of the uterus at the time of diagnosis, it can usually be cured by hysterectomy. Unopposed exposure of endometrial cells to estrogens dramatically increases the chance of developing this form of uterine cancer and it is for this reason that hormone replacement therapy consisting solely of estrogen should not be given to women with intact uteri. Cyclical or continuous co-administration of progesterone serves to prevent excessive proliferation of endometrial cells, reducing the risk of endometrial cancer in post-menopausal women receiving estrogen as part of hormone replacement therapy regimens.
The majority of cases of endometrial cancers express estrogen receptor and, in general, estrogen responsive tumors have a favorable prognosis. Acquired (somatic) mutations have been described in up to 8.5% of cases, however the role of these mutations in the development and progression of endometrial cancer is uncertain at present.
Although it remains somewhat controversial, studies suggest that use of tamoxifen may increase the chance of developing endometrial cancer. This may be because, in addition to its role in estrogen receptor blockade, tamoxifen has partial receptor agonist activity and results in low-grade induction of estrogen responsive genes that induce endometrial proliferation.
Given the involvement of the estrogen receptor in mediating/modulating various disorders, it is critical to identify sequence polymorphisms in the estrogen receptor and to correlate these with disease states, therapeutic effectiveness and the like. The present invention advances the art by providing a variety of previously unidentified polymorphisms in the ESR-beta protein.
SNPs
The genomes of all organisms undergo spontaneous mutation in the course of their continuing evolution, generating variant forms of progenitor sequences (Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)). The variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form or may be neutral. In some instances, a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism. In other instances, a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species and effectively becomes the progenitor form. Additionally, the effect of a variant form may be both beneficial and detrimental, depending on the circumstances. For example, a heterozygous sickle cell mutation confers resistance to malaria, but a homozygous sickle cell mutation is usually lethal. In many instances, both progenitor and variant form(s) survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymorphisms, such as SNPs.
The reference allelic form is arbitrarily designated and may be, for example, the most abundant form in a population, or the first allelic form to be identified, and other allelic forms are designated as alternative, variant or polymorphic alleles. The allelic form occurring most frequently in a selected population is sometimes referred to as the xe2x80x9cwild typexe2x80x9d form.
Approximately 90% of all polymorphisms in the human genome are single nucleotide polymorphisms (SNPs). SNPs are single base pair positions in DNA at which different alleles, or alternative nucleotides, exist in some population. The SNP position, or SNP site, is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations). An individual may be homozygous or heterozygous for an allele at each SNP position. As defined by the present invention, the least frequent allele at a SNP position can have any frequency that is less than the frequency of the more frequent allele, including a frequency of less than 1% in a population. A SNP can, in some instances, be referred to as a xe2x80x9ccSNPxe2x80x9d to denote that the nucleotide sequence containing the SNP is an amino acid coding sequence.
A SNP may arise due to a substitution of one nucleotide for another at the polymorphic site. Substitutions can be transitions or transversions. A transition is the replacement of one purine nucleotide by another purine nucleotide, or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine by a pyrimidine, or vice versa. A SNP may also be a single base insertion/deletion variant (referred to as xe2x80x9cindelsxe2x80x9d). A substitution that changes a codon coding for one amino acid to a codon coding for a different amino acid is referred to as a non-synonymous codon change, or missense mutation. A synonymous codon change, or silent mutation, is one that does not result in a change of amino acid due to the degeneracy of the genetic code. A nonsense mutation is a type of non-synonymous codon change that results in the formation of a stop codon, thereby leading to premature termination of a polypeptide chain and a defective protein.
SNPs, in principle, can be bi-, tri-, or tetra-allelic. However, tri- and tetra-allelic polymorphisms are extremely rare, almost to the point of non-existence (Brookes, Gene 234 (1999) 177-186). For this reason, SNPs are often referred to as xe2x80x9cbi-allelic markersxe2x80x9d, or xe2x80x9cdi-allelic markersxe2x80x9d.
Causative SNPs are those SNPs that produce alterations in gene expression or in the expression or function of a gene product, and therefore are most predictive of a possible clinical phenotype. One such class includes SNPs falling within regions of genes encoding a polypeptide product, i.e. cSNPs. These SNPs may result in an alteration of the amino acid sequence of the polypeptide product (i.e., non-synonymous codon changes) and give rise to the expression of a defective or other variant protein. Furthermore, in the case of nonsense mutations, a SNP may lead to premature termination of a polypeptide product. Such variant products can result in a pathological condition, e.g., genetic disease. Examples of genes in which a polymorphism within a coding sequence gives rise to genetic disease include sickle cell anemia and cystic fibrosis. Causative SNPs do not necessarily have to occur in coding regions; causative SNPs can occur in any region that can ultimately affect the expression and/or activity of the protein encoded by the nucleic acid. Such gene areas include those involved in transcription, such as SNPs in promoter regions, in gene areas involved in transcript processing, such as SNPs at intron-exon boundaries that may cause defective splicing, or SNPs in mRNA processing signal sequences such as polyadenylation signal regions. For example, a SNP may inhibit splicing of an intron and result in mRNA containing a premature stop codon, leading to a defective protein. Consequently, SNPs in regulatory regions can have substantial phenotypic impact.
Some SNPs that are not causative SNPs nevertheless are in close association with, and therefore segregate with, a disease-causing sequence. In this situation, the presence of the SNP correlates with the presence of, or susceptibility to, the disease. These SNPs are invaluable for diagnostics and disease susceptibility screening.
Clinical trials have shown that patient response to treatment with pharmaceuticals is often heterogeneous. Thus there is a need for improved approaches to pharmaceutical agent design and therapy. SNPs can be used to help identify patients most suited to therapy with particular pharmaceutical agents (this is often termed xe2x80x9cpharmacogenomicsxe2x80x9d). Pharmacogenomics can also be used in pharmaceutical research to assist the drug selection process. (Linder et al. (1997), Clinical Chemistry, 43, 254; Marshall (1997), Nature Biotechnology, 15, 1249; International Patent Application WO 97/40462, Spectra Biomedical; and Schafer et al. (1998), Nature Biotechnology, 16, 3.).
Population Studies
Population Genetics is the study of how Mendel""s laws and other genetic principles apply to entire. Such a study is essential to a proper understanding of evolution because, fundamentally, evolution is the result of progressive change in the genetic composition of a population. Population genetics thus seeks to understand and to predict the effects of such genetic phenomena as segregation, recombination, and mutation; at the same time, population genetics must take into account such ecological and evolutionary factors as population size, patterns of mating, geographic distribution of individuals, migration and natural selection.
Ideally, one would wish to know how to describe the types and frequencies of genes in a population, to explain how the population""s genetic composition came to be the way it is, and to predict how the population would change as a result of natural selection or as a result of artificial selection.
In order to explain many of those issues it is important to understand the existing relation between loci denominated: Linkage.
Linkage is the coinheritance of two or more nonallelic genes because their loci are in close proximity on the same chromosome, such that after meiosis they remain associated more often than the 50% expected for unlinked genes. During meiosis, there is a physical crossing over, it is clear that during the production of germ cells there is a physical exchange of maternal and paternal genetic contributions between individual chromatids. This exchange necessarily separates genes in chromosomal regions that were contiguous in each parent and, by mixing them with retained linear order, results in xe2x80x9crecombinantsxe2x80x9d. The process of forming recombinants through meiotic crossing-over is an essential feature in the reassortment of genetic traits and is central to understanding the transmission of genes.
Recombination generally occurs between large segments of DNA. This means that contiguous stretches of DNA and genes are likely to be moved together. Conversely, regions of the DNA that are far apart on a given chromosome are likely to become separated during the process of crossing-over.
It is possible to use molecular markers to clarify the recombination events that take place during meiosis. Some markers as (CA)n repeats of different lengths are dispersed throughout human DNA and there is little selective pressure in their lengths are used as position markers and regional identifying characters along chromosomes. Those markers can be used to distinguished paternally derived from maternally derived gene regions.
Other markers are Single Nucleotide Polymorphism (SNP), those are biallelic markers, also used to analyzed the transmission of those markers to offspring.
The pattern of a set of markers along a chromosome is referred to as a xe2x80x9cHaplotypexe2x80x9d. Therefore sets of alleles on the same small chromosmal segment tend to be transmitted as a block through a pedigree. By analyzing the haplotypes in a series of offspring of parents whose haplotypes are known, it is possible to establish which parental segment of which chromosome was transmitted to which child. When not broken up by recombinations, haplotypes can be treated for mapping purposes as alleles at a single highly polymorphic locus.
The existence of a preferential occurrence of a disease gene in association with specific alleles of linked markers is called xe2x80x9cLinkage Disequilibriumxe2x80x9d (LD). This sort of disequilibrium generally implies that most of the disease chromosomes carry the same mutation and the markers being tested are quite close to the disease gene. For example, there is considerable linkage disequilibrium across the entire HLA locus. The A3 allele is in LD with the B7 and B14 alleles, and as a result B7 and B14 are also highly associated with hemochromatosis. Thus, HLA typing alone can significantly alter the estimate of risk for hemochromatosis, even if other family members are not available for formal linkage analysis. As a result, using a combination of several markers surrounding the presumptive location of the gene, a haplotype can be determined for affected and unaffected family members.
SNP-Based Association Analysis and Linkage Disequilibrium Mapping
SNPs are useful in association studies for identifying particular SNPs, or other polymorphisms, associated with pathological conditions, such as breast cancer. Association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families (linkage studies). An association study using SNPs involves determining the frequency of the SNP allele in many patients with the disorder of interest, such as breast cancer, as well as controls of similar age and race. The appropriate selection of patients and controls is critical to the success of SNP association studies. Therefore, a pool of individuals with well-characterized phenotypes is extremely desirable. For example, blood pressure and heart rate can be correlated with SNP patterns in hypertensive individuals in whom these physiological parameters are known in order to find associations between particular SNP genotypes and known phenotypes. Significant associations between particular SNPs or SNP haplotypes and phenotypic characteristics can be determined by standard statistical methods. Association analysis can either be direct or LD based. In direct association analysis, causative SNPs are tested that are candidates for the pathogenic sequence itself
In LD based SNP association analysis, random SNPs are tested over a large genomic region, possibly the entire genome, in order to find a SNP in LD with the true pathogenic sequence or pathogenic SNP. For this approach, high density SNP maps are required in order for random SNPs to be located close enough to an unknown pathogenic locus to be in linkage disequilibrium with that locus in order to detect an association. SNPs tend to occur with great frequency and are spaced uniformly throughout the genome. The frequency and uniformity of SNPs means that there is a greater probability, compared with other types of polymorphisms such as tandem repeat polymorphisms, that a SNP will be found in close proximity to a genetic locus of interest. SNPs are also mutationally more stable than tandem repeat polymorphisms, such as VNTRs. LD-based association studies are capable of finding a disease susceptibility gene without any a priori assumptions about what or where the gene is.
Currently, however, it is not feasible to do SNP association studies over the entire human genome, therefore candidate genes associated with breast cancer are targeted for SNP identification and association analysis. The candidate gene approach uses a priori knowledge of disease pathogenesis to identify genes that are hypothesized to directly influence development of the disease. The candidate gene approach may focus on a gene that is directly targeted by a drug used to treat the disorder. To discover SNPs associated with an increased susceptibility to breast cancer candidate genes can be selected from systems physiologically implicated in the disease pathway. SNPs found in these genes are then tested for statistical association with disease in individuals who have the disease compared with appropriate controls. The candidate gene approach has the advantages of drastically reducing the number of candidate SNPs, and the number of individuals, that need to be typed, compared with LD-based association studies of random SNPs over large areas of, or complete, genomes. Furthermore, in the candidate gene approach, no assumptions are made about the extent of LD over any particular area of the genome.
Combined with the use of a high density map of appropriately spaced, sufficiently informative SNP markers, association studies, including linkage disequilibrium-based genome wide association studies, will enable the identification of most genes involved in complex disorders, such as cardiovascular diseases, cancer etc. This will enhance the selection of candidate genes most likely to contain causative SNPs associated with a particular disease. All of the SNPs disclosed by the present invention can be employed as part of genome-wide association studies or as part of candidate gene association studies. The present invention advances the state of the art and provides commercially useful embodiments by providing previously unidentified SNPs in the estrogen receptor genes.
The present invention is based on sequencing genomic DNA from human chromosome 6 and cDNAs to define the genomic structure of estrogen receptor beta genes, novel polymorphisms in the estrogen receptor gene/protein. Such polymorphisms can lead to a variety of disorders that are mediated/modulated by a variant estrogen receptor, such as a susceptibility to cancer, osteoporosis, cardiovascular disorders, etc. Based on this sequencing approach, the present invention provides genomic nucleotide sequences, cDNA sequences, amino acid sequences, sequence polymorphisms in the ESR-beta gene, methods of detecting these sequences/polymorphisms in a sample, methods of determining a risk of having or developing a disorder mediated by a variant estrogen receptor and methods of screening for compounds used to treat disorders mediated by a variant estrogen receptor.